Molecular Characteristics and Biological Functions of Surface-Active and Surfactant Proteins.

Many critical biological processes take place at hydrophobic:hydrophilic interfaces, and a wide range of organisms produce surface-active proteins and peptides that reduce surface and interfacial tension and mediate growth and development at these boundaries. Microorganisms produce both small lipid-associated peptides and amphipathic proteins that allow growth across water:air boundaries, attachment to surfaces, predation, and improved bioavailability of hydrophobic substrates. Higher-order organisms produce surface-active proteins with a wide variety of functions, including the provision of protective foam environments for vulnerable reproductive stages, evaporative cooling, and gas exchange across airway membranes. In general, the biological functions supported by these diverse polypeptides require them to have an amphipathic nature, and this is achieved by a diverse range of molecular structures, with some proteins undergoing significant conformational change or intermolecular association to generate the structures that are surface active.

Dynamin 1xA interacts with Endophilin A1 via its spliced long C-terminus for ultrafast endocytosis.

Dynamin 1 mediates fission of endocytic synaptic vesicles in the brain and has two major splice variants, Dyn1xA and Dyn1xB, which are nearly identical apart from the extended C-terminal region of Dyn1xA. Despite a similar set of binding partners, only Dyn1xA is enriched at endocytic zones and accelerates vesicle fission during ultrafast endocytosis. Here, we report that Dyn1xA achieves this localization by preferentially binding to Endophilin A1 through a newly defined binding site within its long C-terminal tail extension. Endophilin A1 binds this site at higher affinity than the previously reported site, and the affinity is determined by amino acids within the Dyn1xA tail but outside the binding site. This interaction is regulated by the phosphorylation state of two serine residues specific to the Dyn1xA variant. Dyn1xA and Endophilin A1 colocalize in patches near the active zone, and mutations disrupting Endophilin A binding to the long tail cause Dyn1xA mislocalization and stalled endocytic pits on the plasma membrane during ultrafast endocytosis. Together, these data suggest that the specificity for ultrafast endocytosis is defined by the phosphorylation-regulated interaction of Endophilin A1 with the C-terminal extension of Dyn1xA.

A Biochemical and Biophysical Analysis of the Interaction of nsp9 with nsp12 from SARS-CoV-2-Implications for Future Drug Discovery Efforts.

The ongoing global pandemic of the coronavirus 2019 (COVID-19) disease is caused by the virus SARS-CoV-2, with very few highly effective antiviral treatments currently available. The machinery responsible for the replication and transcription of viral RNA during infection is made up of several important proteins. Two of these are nsp12, the catalytic subunit of the viral polymerase, and nsp9, a cofactor of nsp12 involved in the capping and priming of viral RNA. While several recent studies have determined the structural details of the interaction of nsp9 with nsp12 in the context of RNA capping, very few biochemical or biophysical details are currently available. In this study, we have used a combination of surface plasmon resonance (SPR) experiments, size exclusion chromatography (SEC) experiments, and biochemical assays to identify specific nsp9 residues that are critical for nsp12 binding as well as RNAylation, both of which are essential for the RNA capping process. Our data indicate that nsp9 dimerization is unlikely to play a significant functional role in the virus. We confirm that a set of recently discovered antiviral peptides inhibit nsp9-nsp12 interaction by specifically binding to nsp9; however, we find that these peptides do not impact RNAylation. In summary, our results have important implications for future drug discovery efforts to combat SARS-CoV-2 and any newly emerging coronaviruses.

Surface-Induced Hydrophobin Assemblies with Versatile Properties and Distinct Underlying Structures.

Hydrophobins are remarkable proteins due to their ability to self-assemble into amphipathic coatings that reverse surface wettability. Here, the versatility of the Class I hydrophobins EASΔ15 and DewY in diverse nanosuspension and coating applications is demonstrated. The hydrophobins are shown to coat or emulsify a range of substrates including oil, hydrophobic drugs, and nanodiamonds and alter their solution and surface behavior. Surprisingly, while the coatings confer new properties, only a subset is found to be resistant to hot detergent treatment, a feature previously thought to be characteristic of the functional amyloid form of Class I hydrophobins. These results demonstrate that substrate surface properties can influence the molecular structures and physiochemical properties of hydrophobin and possibly other functional amyloids. Functional amyloid assembly with different substrates and conditions may be analogous to the propagation of different polymorphs of disease-associated amyloid fibrils with distinct structures, stability, and clinical phenotypes. Given that amyloid formation is not required for Class I hydrophobins to serve diverse applications, our findings open up new opportunities for their use in applications requiring a range of chemical and physical properties. In hydrophobin nanotechnological applications where high stability of assemblies is required, simultaneous structural and functional characterization should be carried out. Finally, while results in this study pertain to synthetic substrates, they raise the possibility that at least some members of the pseudo-Class I and Class III hydrophobins, reported to form assemblies with noncanonical properties, may be Class I hydrophobins adopting alternative structures in response to environmental cues.

A distinct ssDNA/RNA binding interface in the Nsp9 protein from SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel, highly infectious RNA virus that belongs to the coronavirus family. Replication of the viral genome is a fundamental step in the virus life cycle and SARS-CoV-2 non-structural protein 9 (Nsp9) is shown to be essential for virus replication through its ability to bind RNA in the closely related SARS-CoV-1 strain. Two recent studies revealing the three-dimensional structure of Nsp9 from SARS-CoV-2 have demonstrated a high degree of similarity between Nsp9 proteins within the coronavirus family. However, the binding affinity to RNA is very low which, until now, has prevented the determination of the structural details of this interaction. In this study, we have utilized nuclear magnetic resonance spectroscopy (NMR) in combination with surface biolayer interferometry (BLI) to reveal a distinct binding interface for both ssDNA and RNA that is different to the one proposed in the recently solved SARS-CoV-2 replication and transcription complex (RTC) structure. Based on these data, we have proposed a structural model of a Nsp9-RNA complex, shedding light on the molecular details of these important interactions.

Expression and purification of the NG domain from human SRα, a key component of the Signal Recognition Particle (SRP) receptor.

The Signal Recognition Particle (SRP) and the SRP receptor (SR) are responsible for protein targeting to the plasma membrane and the protein secretory pathway. Eukaryotic SRα, one of the two proteins that form the SR, is composed of the NG, MoRF and X domains. The SRα-NG domain is responsible for binding to SRP proteins such as SRP54, interacting with RNA, binding and hydrolysing GTP. The ability to produce folded SRα-NG is a prerequisite for structural studies directed towards a better understanding of its molecular mechanism and function, as well as in (counter-)screening assays for potential binders in the drug development pipeline. However, previously reported SRα-NG constructs and purification methods only used a truncated version, lacking the first N-terminal helix. This helix in other NG domains (e.g., SRP54) has been shown to be important for protein:protein interactions but its importance in SRα remains unknown. Here, we present the cloning as well as optimised expression and purification protocols of the whole SRα-NG domain including the first N-terminal helix. We have also expressed and purified isotopically labelled SRα-NG to facilitate Nuclear Magnetic Resonance (NMR) studies.

Hydrophobin Rodlets on the Fungal Cell Wall.

The conidia of airborne fungi are protected by a hydrophobic protein layer that coats the cell wall polysaccharides and renders the spores resistant to wetting and desiccation. A similar layer is presented on the outer surface of the aerial hyphae of some fungi. This layer serves multiple purposes, including facilitating spore dispersal, mediating the growth of hyphae into the air from moist environments, aiding host interactions in symbiotic relationships and increasing infectivity in pathogenic fungi. The layer consists of tightly packed, fibrillar structures termed "rodlets", which are approximately 10 nm in diameter, hundreds of nanometres long and grouped in fascicles. Rodlets are an extremely stable protein structure, being resistant to detergents, denaturants and alcohols and requiring strong acids for depolymerisation. They are produced through the self-assembly of small, surface-active proteins that belong to the hydrophobin protein family. These small proteins are expressed by all filamentous fungi and are characterised by a high proportion of hydrophobic residues and the presence of eight cysteine residues. Rodlets are a form of the functional amyloid fibril, where the hydrophobin monomers are held together in the rodlets by intermolecular hydrogen bonds that contribute to a stable ß-sheet core.

Disparate binding kinetics by an intrinsically disordered domain enables temporal regulation of transcriptional complex formation.

Intrinsically disordered regions are highly represented among mammalian transcription factors, where they often contribute to the formation of multiprotein complexes that regulate gene expression. An example of this occurs with LIM-homeodomain (LIM-HD) proteins in the developing spinal cord. The LIM-HD protein LHX3 and the LIM-HD cofactor LDB1 form a binary complex that gives rise to interneurons, whereas in adjacent cell populations, LHX3 and LDB1 form a rearranged ternary complex with the LIM-HD protein ISL1, resulting in motor neurons. The protein-protein interactions within these complexes are mediated by ordered LIM domains in the LIM-HD proteins and intrinsically disordered LIM interaction domains (LIDs) in LDB1 and ISL1; however, little is known about how the strength or rates of binding contribute to complex assemblies. We have measured the interactions of LIM:LID complexes using FRET-based protein-protein interaction studies and EMSAs and used these data to model population distributions of complexes. The protein-protein interactions within the ternary complexes are much weaker than those in the binary complex, yet surprisingly slow LDB1:ISL1 dissociation kinetics and a substantial increase in DNA binding affinity promote formation of the ternary complex over the binary complex in motor neurons. We have used mutational and protein engineering approaches to show that allostery and modular binding by tandem LIM domains contribute to the LDB1LID binding kinetics. The data indicate that a single intrinsically disordered region can achieve highly disparate binding kinetics, which may provide a mechanism to regulate the timing of transcriptional complex assembly.

The BRD3 ET domain recognizes a short peptide motif through a mechanism that is conserved across chromatin remodelers and transcriptional regulators.

Members of the bromodomain and extra-terminal domain (BET) family of proteins (bromodomain-containing (BRD) 2, 3, 4, and T) are widely expressed and highly conserved regulators of gene expression in eukaryotes. These proteins have been intimately linked to human disease, and more than a dozen clinical trials are currently underway to test BET-protein inhibitors as modulators of cancer. However, although it is clear that these proteins use their bromodomains to bind both histones and transcription factors bearing acetylated lysine residues, the molecular mechanisms by which BET family proteins regulate gene expression are not well defined. In particular, the functions of the other domains such as the ET domain have been less extensively studied. Here, we examine the properties of the ET domain of BRD3 as a protein/protein interaction module. Using a combination of pulldown and biophysical assays, we demonstrate that BRD3 binds to a range of chromatin-remodeling complexes, including the NuRD, BAF, and INO80 complexes, via a short linear "KIKL" motif in one of the complex subunits. NMR-based structural analysis revealed that, surprisingly, this mode of interaction is shared by the AF9 and ENL transcriptional coregulators that contain an acetyl-lysine-binding YEATS domain and regulate transcriptional elongation. This observation establishes a functional commonality between these two families of cancer-related transcriptional regulators. In summary, our data provide insight into the mechanisms by which BET family proteins might link chromatin acetylation to transcriptional outcomes and uncover an unexpected functional similarity between BET and YEATS family proteins.

Expression, purification and crystallization of human kynurenine aminotransferase 2 exploiting a highly optimized codon set.

Kynurenine aminotransferase (KAT) is a pyridoxal-5'-phosphate (PLP) dependent enzyme that catalyses kynurenine (KYN) to kynurenic acid (KYNA), a neuroactive product in the tryptophan metabolic pathway. Evidence suggests that abnormal levels of KYNA are involved in many neurodegenerative diseases such as Parkinson's disease, Huntington's disease, Alzheimer's disease and schizophrenia. Reducing KYNA production through inhibiting kynurenine aminotransferase 2 (KAT2) would be a promising approach to understanding and treating the related neurological and mental disorders. In this study we used an optimized codon sequence to overexpress histidine-tagged human KAT2 (hKAT2) using an Escherichia coli expression system. After a single step of Ni-NTA based purification the purified protein (>95%) was confirmed to be active by an HPLC based activity assay and was crystallized using the hanging-drop vapour diffusion method. The crystal system represents a novel space group, and a complete X-ray diffraction data set was collected to 1.83 Å resolution, and higher resolution data than for any reported native human KAT2 structure. The optimised method of protein production provides a fast and reliable technique to generate large quantities of active human KAT2 suitable for future small-molecule lead compound screening and structural design work.

Self-assembly of functional, amphipathic amyloid monolayers by the fungal hydrophobin EAS.

The hydrophobin EAS from the fungus Neurospora crassa forms functional amyloid fibrils called rodlets that facilitate spore formation and dispersal. Self-assembly of EAS into fibrillar rodlets occurs spontaneously at hydrophobic:hydrophilic interfaces and the rodlets further associate laterally to form amphipathic monolayers. We have used site-directed mutagenesis and peptide experiments to identify the region of EAS that drives intermolecular association and formation of the cross-ß rodlet structure. Transplanting this region into a nonamyloidogenic hydrophobin enables it to form rodlets. We have also determined the structure and dynamics of an EAS variant with reduced rodlet-forming ability. Taken together, these data allow us to pinpoint the conformational changes that take place when hydrophobins self-assemble at an interface and to propose a model for the amphipathic EAS rodlet structure.

Intrinsic disorder modulates protein self-assembly and aggregation.

Protein molecules have evolved to adopt distinctive and well-defined functional and soluble states under physiological conditions. In some circumstances, however, proteins can self-assemble into fibrillar aggregates designated as amyloid fibrils. In vivo these processes are normally associated with severe pathological conditions but can sometimes have functional relevance. One such example is the hydrophobins, whose aggregation at air-water interfaces serves to create robust protein coats that help fungal spores to resist wetting and thus facilitate their dispersal in the air. We have performed multiscale simulations to address the molecular determinants governing the formation of functional amyloids by the class I fungal hydrophobin EAS. Extensive samplings of full-atom replica-exchange molecular dynamics and coarse-grained simulations have allowed us to identify factors that distinguish aggregation-prone from highly soluble states of EAS. As a result of unfavourable entropic terms, highly dynamical regions are shown to exert a crucial influence on the propensity of the protein to aggregate under different conditions. More generally, our findings suggest a key role that specific flexible structural elements can play to ensure the existence of soluble and functional states of proteins under physiological conditions.

A structural basis for the regulation of the LIM-homeodomain protein islet 1 (Isl1) by intra- and intermolecular interactions.

Islet 1 (Isl1) is a transcription factor of the LIM-homeodomain (LIM-HD) protein family and is essential for many developmental processes. LIM-HD proteins all contain two protein-interacting LIM domains, a DNA-binding homeodomain (HD), and a C-terminal region. In Isl1, the C-terminal region also contains the LIM homeobox 3 (Lhx3)-binding domain (LBD), which interacts with the LIM domains of Lhx3. The LIM domains of Isl1 have been implicated in inhibition of DNA binding potentially through an intramolecular interaction with or close to the HD. Here we investigate the LBD as a candidate intramolecular interaction domain. Competitive yeast-two hybrid experiments indicate that the LIM domains and LBD from Isl1 can interact with apparently low affinity, consistent with no detection of an intermolecular interaction in the same system. Nuclear magnetic resonance studies show that the interaction is specific, whereas substitution of the LBD with peptides of the same amino acid composition but different sequence is not specific. We solved the crystal structure of a similar but higher affinity complex between the LIM domains of Isl1 and the LIM interaction domain from the LIM-HD cofactor protein LIM domain-binding protein 1 (Ldb1) and used these coordinates to generate a homology model of the intramolecular interaction that indicates poorer complementarity for the weak intramolecular interaction. The intramolecular interaction in Isl1 may provide protection against aggregation, minimize unproductive DNA binding, and facilitate cofactor exchange within the cell.

New insights into DNA recognition by zinc fingers revealed by structural analysis of the oncoprotein ZNF217.

Classical zinc fingers (ZFs) are one of the most abundant and best characterized DNA-binding domains. Typically, tandem arrays of three or more ZFs bind DNA target sequences with high affinity and specificity, and the mode of DNA recognition is sufficiently well understood that tailor-made ZF-based DNA-binding proteins can be engineered. We have shown previously that a two-zinc finger unit found in the transcriptional coregulator ZNF217 recognizes DNA but with an affinity and specificity that is lower than other ZF arrays. To investigate the basis for these differences, we determined the structure of a ZNF217-DNA complex. We show that although the overall position of the ZFs on the DNA closely resembles that observed for other ZFs, the side-chain interaction pattern differs substantially from the canonical model. The structure also reveals the presence of two methyl-π interactions, each featuring a tyrosine contacting a thymine methyl group. To our knowledge, interactions of this type have not previously been described in classical ZF-DNA complexes. Finally, we investigated the sequence specificity of this two-ZF unit and discuss how ZNF217 might discriminate its target DNA sites in the cell.

A structural analysis of DNA binding by myelin transcription factor 1 double zinc fingers.

Myelin transcription factor 1 (MyT1/NZF2), a member of the neural zinc-finger (NZF) protein family, is a transcription factor that plays a central role in the developing central nervous system. It has also recently been shown that, in combination with two other transcription factors, the highly similar paralog MyT1L is able to direct the differentiation of murine and human stem cells into functional neurons. MyT1 contains seven zinc fingers (ZFs) that are highly conserved throughout the protein and throughout the NZF family. We recently presented a model for the interaction of the fifth ZF of MyT1 with a DNA sequence derived from the promoter of the retinoic acid receptor (RARE) gene. Here, we have used NMR spectroscopy, in combination with surface plasmon resonance and data-driven molecular docking, to delineate the mechanism of DNA binding for double ZF polypeptides derived from MyT1. Our data indicate that a two-ZF unit interacts with the major groove of the entire RARE motif and that both fingers bind in an identical manner and with overall two-fold rotational symmetry, consistent with the palindromic nature of the target DNA. Several key residues located in one of the irregular loops of the ZFs are utilized to achieve specific binding. Analysis of the human and mouse genomes based on our structural data reveals three putative MyT1 target genes involved in neuronal development.

Solution structure and interface-driven self-assembly of NC2, a new member of the Class II hydrophobin proteins.

Hydrophobins are fungal proteins that self-assemble spontaneously to form amphipathic monolayers at hydrophobic:hydrophilic interfaces. Hydrophobin assemblies facilitate fungal transitions between wet and dry environments and interactions with plant and animal hosts. NC2 is a previously uncharacterized hydrophobin from Neurospora crassa. It is a highly surface active protein and is able to form protein layers on a water:air interface that stabilize air bubbles. On a hydrophobic substrate, NC2 forms layers consisting of an ordered network of protein molecules, which dramatically decrease the water contact angle. The solution structure and dynamics of NC2 have been determined using nuclear magnetic resonance spectroscopy. The structure of this protein displays the same core fold as observed in other hydrophobin structures determined to date, including the Class II hydrophobins HFBI and HFBII from Trichoderma reesei, but certain features illuminate the structural differences between Classes I and II hydrophobins and also highlight the variations between structures of Class II hydrophobin family members. The unique properties of hydrophobins have attracted much attention for biotechnology applications. The insights obtained through determining the structure, biophysical properties and assembly characteristics of NC2 will facilitate the development of hydrophobin-based applications.

Solid-state NMR structure determination from diagonal-compensated, sparsely nonuniform-sampled 4D proton-proton restraints.

We report acquisition of diagonal-compensated protein structural restraints from four-dimensional solid-state NMR spectra on extensively deuterated and (1)H back-exchanged proteins. To achieve this, we use homonuclear (1)H-(1)H correlations with diagonal suppression and nonuniform sampling (NUS). Suppression of the diagonal allows the accurate identification of cross-peaks which are otherwise obscured by the strong autocorrelation or whose intensity is biased due to partial overlap with the diagonal. The approach results in unambiguous spectral interpretation and relatively few but reliable restraints for structure calculation. In addition, the diagonal suppression produces a spectrum with low dynamic range for which ultrasparse NUS data sets can be readily reconstructed, allowing straightforward application of NUS with only 2% sampling density with the advantage of more heavily sampling time-domain regions of high signal intensity. The method is demonstrated here for two proteins, α-spectrin SH3 microcrystals and hydrophobin functional amyloids. For the case of SH3, suppression of the diagonal results in facilitated identification of unambiguous restraints and improvement of the quality of the calculated structural ensemble compared to nondiagonal-suppressed 4D spectra. For the only partly assigned hydrophobin rodlets, the structure is yet unknown. Applied to this protein of biological significance with large inhomogeneous broadening, the method allows identification of unambiguous crosspeaks that are otherwise obscured by the diagonal.

Engineering specificity changes on a RanBP2 zinc finger that binds single-stranded RNA.

The realization that gene transcription is much more pervasive than previously thought and that many diverse RNA species exist in simple as well as complex organisms has triggered efforts to develop functionalized RNA-binding proteins (RBPs) that have the ability to probe and manipulate RNA function. Previously, we showed that the RanBP2-type zinc finger (ZF) domain is a good candidate for an addressable single-stranded-RNA (ssRNA) binding domain that can recognize ssRNA in a modular and specific manner. In the present study, we successfully engineered a sequence specificity change onto this ZF scaffold by using a combinatorial approach based on phage display. This work constitutes a foundation from which a set of RanBP2 ZFs might be developed that is able to recognize any given RNA sequence.

The motif of human cardiac myosin-binding protein C is required for its Ca2+-dependent interaction with calmodulin.

The N-terminal modules of cardiac myosin-binding protein C (cMyBP-C) play a regulatory role in mediating interactions between myosin and actin during heart muscle contraction. The so-called "motif," located between the second and third immunoglobulin modules of the cardiac isoform, is believed to modulate contractility via an "on-off" phosphorylation-dependent tether to myosin ΔS2. Here we report a novel Ca(2+)-dependent interaction between the motif and calmodulin (CaM) based on the results of a combined fluorescence, NMR, and light and x-ray scattering study. We show that constructs of cMyBP-C containing the motif bind to Ca(2+)/CaM with a moderate affinity (K(D) ∼10 µM), which is similar to the affinity previously determined for myosin ΔS2. However, unlike the interaction with myosin ΔS2, the Ca(2+)/CaM interaction is unaffected by substitution with a triphosphorylated motif mimic. Further, Ca(2+)/CaM interacts with the highly conserved residues (Glu(319)-Lys(341)) toward the C-terminal end of the motif. Consistent with the Ca(2+) dependence, the binding of CaM to the motif is mediated via the hydrophobic clefts within the N- and C-lobes that are known to become more exposed upon Ca(2+) binding. Overall, Ca(2+)/CaM engages with the motif in an extended clamp configuration as opposed to the collapsed binding mode often observed in other CaM-protein interactions. Our results suggest that CaM may act as a structural conduit that links cMyBP-C with Ca(2+) signaling pathways to help coordinate phosphorylation events and synchronize the multiple interactions between cMyBP-C, myosin, and actin during the heart muscle contraction.